The present invention relates generally to derivatized solid supports. More particularly, the present invention relates to solid supports derivatized with silanes containing aldehydic functionalities and their use in biological applications.
The use of immobilized bio-molecules is an essential technique required for many biological applications. A common method used to immobilize bio-molecules is by reaction of the primary amine groups of the biological molecules with an aldehyde functionality that is bonded to a solid support matrix.
A popular method of introducing aldehydes to the solid support matrix is through the activation of an amine-functionalized surface with a glutaraldehyde solution. Although popular, this method has several disadvantages. First glutaraldehyde is an unstable compound that is difficult to purify. Additionally, two Schiff bases are present in the covalent linkage of the bio-molecule to the support. Additionally, the Schiff base linkage of the glutaraldehyde to the support is susceptible to hydrolysis and thus may lead to ligand leaching. Treatment with a reducing agent such as sodium borohydride or sodium cyanoborohydride to remove the Schiff bases can be performed, but this adds an additional step to the process.
There remains a need for a solid support matrix with bonded aldehydic functionalities for immobilizing bio-molecules that is stable and can be produced in a simple process.
Accordingly, it is desirable to provide a solid support matrix containing bonded aldehydic functionalities that is stable and can be used to immobilize bio-molecules. It is further desirable to provide a simple method for producing such a solid support matrix. It is still further desirable to provide an apparatus and method for using a solid support matrix with aldehydic functionalities to immobilize bio-molecules for biological applications.
The present invention is directed to a derivatized solid support matrix containing bonded aldehydic functionalities, that is stable and can be used to immobilize bio-molecules, as well as to a method for producing such a derivatized solid support matrix.
The present invention is further directed to an apparatus and method for using a derivatized solid support matrix with aldehydic functionalities to immobilize bio-molecules for biological applications.
In accordance with one embodiment of the present invention, a method of producing a derivatized matrix material with aldehyde groups is provided. A raw support matrix material having a surface area with hydroxyl groups occurring on the surface area is activated with an acid. The activated support material is then exposed to an alkoxy aldehydic silane to produce a derivatized matrix material. The support matrix material can be selected from a number of materials, including but not limited to glasses, agarose, silica, alumina, glass-coated ELISA plates, resin, nickel, aluminum, zinc and paramagnetic iron. The support matrix material may have naturally occurring hydroxyl groups, or the hydroxyl groups may be introduced artificially. A number of mono, di and tri alkoxy aldehydic silanes may be used for producing a derivatized matrix material with aldehyde groups. The alkoxy aldehydic silane is preferably a trialkoxy aldehydic silane.
In another embodiment of the present inventions, an aldehydic derivatized matrix material comprises a support matrix material having a surface area, and a siloxane coating is disposed on at least a portion of the surface area. The siloxane coating may be a mono-layer or a cross linked siloxane polymer coating. The siloxane coating has a plurality of organic substituents containing aldehydic functional groups pendant therefrom. The organic substituents are bound to the siloxane coating via carbon-silicon covalent bonds.
In accordance with another embodiment of the invention, the aldehydic derivatized support matrix material can be incorporated into an apparatus for immobilizing bio-molecules for biological applications. Such biological applications include, but are not limited to, combinatorial chemistry, molecular biology, ELISA (Enzyme-Linked Immunosorbent Assay) plates, and cell sorting and identification.
There are additional features of the invention that will be described below and which will form the subject matter of the claims appended hereto.
It is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract included below, are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
One aspect of the present invention provides a composition comprising a derivatized solid support matrix material having a surface and a siloxane coating on at least a portion of its surface, linked through Sixe2x80x94O bonds. The siloxane coating comprises a plurality of silicon atoms that are either individually mono-linked to the solid support matrix or cross-linked to each other and further bonded to the support matrix through Sixe2x80x94O bonds to form a siloxane polymer. Each Si unit in the coating has a pendant aldehyde containing organic substituent, bound to the siloxane coating via carbon-silicon covalent bonds. The siloxane coating is further bound to the solid support matrix material through Sixe2x80x94Oxe2x80x94M bonds, wherein M is the support matrix material.
According to one embodiment of the composition, the siloxane coating comprises a multi layer siloxane polymer 1 to 10 Si units deep, preferably 2 to 5 units deep. When viewed in cross section, this embodiment has the general structure: 
where R is an aldehyde containing organic substituent. R1 is C1 to C30 alkyl, C1 to C30 alkenyl, phenyl, naphthyl or hydrogen, and M is the support matrix material. It should be recognized that in this embodiment the placement of the individual Si units is random with respect to the support matrix material. The example given is for illustrative purposes only and is not meant to limit the scope of the invention.
In an alternate embodiment, the individual Si units are cross linked to form a siloxane polymer 1 Si unit deep, which when viewed in cross section, has the structure: 
where R is an aldehyde containing organic substituent and M is the support matrix material.
In a further embodiment, the individual Si units are mono linked to the solid support matrix material, which when viewed in cross section would have the structure: 
where R is an aldehyde containing organic substituent. R1 is alkyl or alkenyl containing 1 to 30 carbon atoms, phenyl, naphthyl or hydrogen and M is the support matrix material.
The aldehyde containing organic substituent, R, may be a straight chain, branched or cyclic alkane containing from 1 to 30 carbon atoms. Alternatively, R may be aromatic or some other unsaturated aldehyde-containing hydrocarbon. For example, wherein R is formaldehyde, a single layer polymer coating would have the following structure when viewed in cross section: 
In an alternative embodiment the aldehydic organic substituents pendant from the Si units may vary between the individual Si units, as shown by: 
where Rxe2x80x2, Rxe2x80x3 and Rxe2x80x2xe2x80x3 may all be different aldehyde containing organic substituents.
Suitable materials for the solid support matrix include, but are not limited to glasses, agarose, silica, alumina, glass-coated ELISA plates, resin, nickel, aluminum, zinc and paramagnetic iron. Preferably, the solid support matrix material comprises silica. The support matrix material may be granular or in the form of beads. The support may also be a glass, metal or ceramic slide, or granular or bead material supported on a glass, metal or ceramic slide.
Another aspect of the current invention provides a method for producing a derivatized matrix support material as described above. In this aspect of the invention, a raw solid support matrix material is provided. The raw solid support matrix material has hydroxyl groups occurring on its surface area. The hydroxyl groups may be naturally occurring or may be artificially introduced by methods well understood by those skilled in the art. Such methods include, but are not limited to treatment with aqueous base, plasma treatments or corona treatments. Preferably, the solid support matrix material is a silica gel.
The hydroxyl groups on the solid support matrix material may be activated using an acid. Preferably, a suspension of the matrix material is formed in an organic solvent and an aqueous acid is added to the suspension. Preferably the organic solvent is a non-polar solvent, such as hexanes or n-heptane. The acidified suspension is then mixed and allowed to equilibrate.
An aldehyde functionality is introduced via an alkoxy aldehydic silane. The alkoxy aldehydic silane may be a single species or mixture of species selected from the group having the general formulas: 
where R1 is an alkyl or alkenyl containing 1 to 30 carbon atoms, a phenyl, a naphthyl, or is a covalent bond. R2, R3 and R4 are independently alkyl or alkenyl containing 1 to 30 carbon atoms, phenyl, naphthyl, silyl or hydrogen. In a preferred embodiment, the alkoxy silane is a trialkoxy silane according to structure 3, and none of R2, R3 or R4 is hydrogen. In a more preferred embodiment, R2, R3 and R4 are equivalent. Preferred R2, R3 and R4 groups are methyl, ethyl and propyl. Preferred R1 groups are straight chain alkanes having 1 to 10 carbon atoms.
The alkoxy silane is added to the acidified suspension and allowed to react with the activated hydroxyl groups on the surface of the matrix material. Preferably, the alkoxy silane is added in small portions over a period of hours. Most preferably, there is an equilibration period between additions of the alkoxy silane. For example, 10 mL of aldehydic alkoxy silane may be added to a suspension of 30 grams of silica in 0.5 mL portions at intervals of 8 to 12 minutes over a period of 3 to 4 hours. Under the acidic conditions in the suspension, the alkoxy silane will be hydrolyzed, producing an alcohol and silanol for each alkoxy substituent, following the general formula:
RSi(ORxe2x80x2)3+3H2Oxe2x86x92RSi(OH)3+3Rxe2x80x2OH 
where R is the aldehyde containing organic substituent. The silanol thus produced can then react with the activated hydroxyl groups on the surface of the matrix material to produce the derivatized material. It will be recognized that the stoichiometry of the reaction will vary depending on alkoxy aldehydic silane employed.
Following addition of the alkoxy silane, the now derivatized matrix material is preferably collected and washed to remove excess acid, unbound silane and polymers.
It is recognized that a number of solid support matrix materials can be used alone, or in combination to achieve a derivatized material having desired qualities. It is also recognized that mixtures of alkoxy silanes having different aldehydic substituents can be used to produce a derivatized material having a wide variety of desired qualities.
The derivatized aldehydic material thus produced can be used as part of an apparatus for immobilizing bio-molecules for biological applications. In one embodiment of this aspect of the invention, a polypropylene column is used to contain an aldehydic derivatized support matrix material according to the current invention. The column is open at one end and has a porous bed support at the opposite end for supporting the matrix material, while allowing fluid to pass. An example of a column suitable for use in this embodiment is the POLY-PREP(copyright) conical polypropylene column, available from Bio-Rad Laboratories Inc.
In another embodiment of this aspect of the invention a column may be used that is compatible for use with a centrifuge. In this embodiment, separation of sample components is affected through centrifugal force. An example of a column suitable for use in this embodiment is the MICRO BIO-SPIN(copyright) chromatography columns, also available from Bio-Rad Laboratories Inc. However, it will be recognized that the invention is not limited to a particular brand of column or a particular design, shape or material of construction.
The apparatus as described in the embodiments above can be used to immobilize a wide variety of bio-molecules. The general procedure followed to immobilize a bio-molecule involves first washing the derivatized support matrix material contained in the column with a buffer solution of appropriate pH. The pH of the buffer solution will vary depending on the bio-molecule to be immobilized and the derivatized support matrix material being used. However, the pH of the buffer solution is preferably in the range of about 4 to about 10, more preferably from about 8 to about 10. A solution containing the bio-molecule to be immobilized is then added to the column in a buffer solution. The apparatus containing the bio-molecule solution is then incubated at an appropriate temperature, for a time sufficient to immobilize at least a portion of the bio-molecule contained in the solution. Again incubation temperatures and times will vary depending on the bio-molecule being immobilized and the derivatized support matrix material being used. Following the incubation period, the column is drained of solution and preferably washed at least once with a fresh buffer solution. Preferably, the buffer solution used is identical to the solution used to initially wash the column. Washing with buffer solution removes any unbound material from the column. The apparatus containing the immobilized bio-molecule may then be further derivatized or used in an assay as desired.
In another embodiment of this aspect of the invention, the support material is a glass coated ELISA (Enzyme-Linked Immunosorbent Assay) plate. In this embodiment, the test bio-molecule is attached covalently to the ELISA plate. A rapid test can then be performed, where an antibody or antigen is coupled to an enzyme as a means for detecting an antigenic match.
Other applications where the current invention may be employed include combinatorial chemistry; tethered amine modified oligonucleotides for PCR (Polymerase Chain Reaction); RNA isolation/purification; DNA micro arrays, probes and genome chips; cell sorting and identification.
The following examples demonstrate the method of producing a derivatized support material according to the current invention and a method of using the invention for immobilizing bio-molecules.